There is a fundamental gap in our current understanding of how DNA replication proceeds through naturally occurring barriers and how sister chromatid cohesion (SCC) is established during DNA replication. During replication of the genome, the replisome complexes encounter a variety of barriers. These include unnatural and natural impediments. Unnatural impediments include DNA lesions and double-strand breaks. Natural impediments include repetitive DNA, DNA-bound proteins and sites of RNA transcription. Based on the size of the human genome, replisome complexes are predicted to stall many times at natural impediments throughout the course of S-phase. Since these natural impediments do not require repair, the cell has evolved mechanisms to prevent these impediments from causing a DNA damage response and cell cycle arrest. However, how this occurs remains poorly understood. The long-term goal of this project is to elucidate how DNA replication proceeds through natural chromosome barriers, such as repetitive DNA sequences and DNA-bound proteins. The objective of this career development award is two-fold: 1) complete the specific aims of the research proposal, which are to determine the non-telomere roles of the CTC1-STN1-TEN1 (CST) complex in DNA replication and SCC, and 2) receive career development through learning new techniques, developing an independent project, gaining teaching experience and receiving guided mentoring in the K99 phase of this award. The K99 phase will occur under the mentorship and guidance of Dr. Carolyn Price at the University of Cincinnati with additional support from Drs. Paul Chastain, David Kaufman, Prasad Jallepalli and Dr. Birgit Ehmer. Natural impediments in our genome that stall replication include difficult-to-replicate DNA regions such as telomeres, fragile sites, trinucleotide repeats and centromeric DNA. At these regions, the replisome must be restarted after stalling. However, how DNA synthesis is reinitiated at these sites remains poorly characterized. SCC is established during DNA replication and proposed to occur concurrent with passage of the replisome. Surprisingly, my preliminary findings suggest that the newly discovered, telomere-associated CST complex not only functions at the telomere but also in both DNA replication restart and SCC. Interestingly, depletion of several other DNA replication proteins leads to defects in SCC and DNA replication restart, suggesting a link between these two processes. Two components of CST, CTC1 and STN1, were originally identified as DNA polymerase alpha- primase (pol alpha) accessory factors, which stimulate pol alpha binding and primase activities. CST also binds ssDNA and is structurally similar to the replication/repair factor replication protein A (RPA). Together, these findings suggest that CST interactions with pol alpha are important for its non-telomere functions. The central hypothesis of this proposal is that CST prevents genome instability by promoting rapid replication restart and SCC at sites of difficult-to replicate DNA, such as telomeres and fragile sites. The proposed research will address this hypothesis through three specific aims: 1) To determine the mechanism by which CST facilitates replication restart after fork stalling; 2) To elucidate the role of CST in sister chroatid cohesion and mitotic progression; 3) To identify CST interactions with replication restart and sister chromatid cohesion factors. In the first aim, the role of CST in replication restart will be investigated by analyzing restart at both the cellular and molecular level in CST-depleted cell lines, determining whether CST is localized to sites of fork stalling, analyzing replication fork stalling in CST-depleted cell lines at sites of difficult-to-replicate DNA and characterizing CST ssDNA binding activity. To perform these experiments, I will receive training in DNA fiber analysis from Dr. Paul Chastain at the University of North Carolina, employ a new protocol for isolating DNA at stalled replication forks and utilize my biochemical and cell biology training. In the second aim, the role of CST in SCC will be assessed by first determining the timing of cohesion loss and whether defects in mitotic progression arise from SCC loss in CST-depleted cells. These studies will require me to learn live-cell imaging and new cell biology techniques. For these studies, I will be collaborating with Dr. Prasad Jallepalli, associate member and laboratory head at the Memorial Sloan-Kettering Cancer Center and an expert in chromosome cohesion and mitosis. The third aim will use a multi-pronged approach to determine CST interacting partners. These studies will include hypothesis-driven experiments to identify CST interactions with proteins involved in DNA replication restart and SCC. CST pull-down followed by mass spectrometry will be used as an unbiased approach to gain insight into CST function through the identification of novel interacting peptides. This proposed work is innovative because: 1) it addresses the unexpected non-telomere functions of CST; 2) it investigates novel mechanisms for the reinitiation of DNA synthesis after fork stalling at natural impediments; 3) it combines a variety of new and well-established techniques to investigate the central hypothesis. The work is significant because it will reveal some of the underlying mechanisms of chromosome instability. Each time a cell divides its DNA must be properly replicated and SCC maintained to ensure proper chromosome segregation to the daughter cells. Defects in either DNA replication or chromosome cohesion lead to phenotypes associated with cancer initiation, such as translocations, deletions, chromosome fusions, gene duplication and aneuploidy. Several genetic disorders, termed cohesionopathies, are also associated with SCC loss and chromosome breakage. Furthermore, mutations in CTC1 were recently shown to underlie a rare autosomal recessive disorder, Coats plus. The completion of these studies will advance our understanding of these cellular processes and provide new targets for prevention and treatment of these diseases.